Alginate layer system for chondrogenic differentiation of human mesenchymal stem cells

ABSTRACT

Disclosed are a composition of chemically defined components which support in vitro and in vivo chondrogenesis of mesenchymal stem cells, a method for in vitro and in vivo chondrogenic induction of such stem cells, and a method of forming human chondrocytes in vitro and in vivo from such stem cells.

This application claims priority based on provisional application SerialNo. 60/108,594, filed Nov. 16. 1998.

The present invention relates to the field of methods and compositionsfor directing human mesenchymal stem cells in vitro and in vivo todifferentiate into chondrocytes prior to or at the time of, or aftertheir implantation into a recipient or host for the therapeutictreatment of articular cartilage defects.

Mesenchymal stem cells (MSCs) are the formative pluripotent blast orembryonic-like cells found in bone marrow, blood, dermis, and periosteumthat are capable of differentiating into specific types of mesenchymalor connective tissues including adipose, osseous, cartilaginous,elastic, muscular, and fibrous connective tissues. The specificdifferentiation pathway which these cells enter depends upon variousinfluences such as mechanical influences and/or endogenous bioactivefactors, including growth factors cytokines and/or localmicroenvironmental conditions established by host tissues.

A clonal rat fetus calvarial cell line has been shown to differentiateinto muscle, fat, cartilage and bone (Goshima et al., Clin Orthop RelRes. 269:274-283, 1991). Bone marrow cells form bone and cartilagefollowing their encasement in diffusion chambers and in vivotransplantation (Ashton et al., Clin Orthop Rel Res. 151:294-307, 1980(rabbit): Bruder et al., Bone Mineral. 11:141-151, 1990 (avian)).Cultured chick periosteum cells have been shown to differentiate intocartilage and bone in vitro (Nakahara et al., Exp. Cell Res.,195:492-503; 1991). Rat bone marrow-derived mesenchymal cells were shownto have the capacity to differentiate into osteoblasts and chondrocyteswhen implanted in vivo (Dennis et al., Cell Transpl, 1:2332, 1991;Goshima et al., Clin Orthop Rel Res. 269:274-283, 1991).

Chondrogenic differentiation of rabbit bone marrow derived mesenchymalprogenitor cells has been studied in connection with articular cartilagehealing utilizing cells in a pelleted format (Johnstone et al. Exp CellRes 238(1):265-272 (1998).). However, cells in a condensed packed orpelleted cell mass do not have an optimal configuration in part due tothe limitation on the maximum growth of the cells, limited permeabilityof nutrients, gases and growth factors, and other metaboliccharacteristics.

Pre-molded biodegradable multilayer matrices have been described forrepair of articular cartilage, which have been packed into orpress-fitted into regularly shaped osteochondral defects (AthanasiouU.S. Pat. No. 5,607,474). Cultured chondrocytes added to a collagenmatrix for implantation into an articular cartilage lesion have alsobeen described (Frenkel, S R et al., J Bone Joint Surg, 79-B(5):831-6(1997)).

Alginate sponges have been used in studies of cartilage repair (seereview Messner K. and J. Gilquist, Acta Orthop. Scand 67(5):523-529(1996)). Mesenchymal cells from 12 day old mouse limb buds that werephenotypically undifferentiated but committed to differentiate to thechondrocytic lineage in an alginate bead culture system differentiatedto cartilage cells and formed a pericellular matrix (Shakibaci, M. andP. De Souza, Cell Biology International. 21(2):75-86 (1997)). Adulthuman chondrocytes cultured in alginate beads formed a compartmentalizedcartilage matrix (Häuselmann H J et al., Am. J. Physiol. 271 (CellPhysiol. 40):C742-C752, 1996). The growth of chondrocytes in alginateand collagen carrier gels has been compared (van Susante, J. et al.,Acta Orthop Scand. 66(6):549-556 (1995)).

An optimized matrix to regenerate cartilage in vivo using mesenchymalstem cells is therefor required.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, there is provided aconstruct which supports the differentiation and maturation of humanmesenchymal stem cells into chondrocytes. In a preferred embodiment, theconstruct comprises human mesenchymal stem cells in association with agel and preferably in an alginate suspension. The construct can beutilized for in vivo cartilage regeneration.

In a further aspect of the invention, soluble hyaluronic acid may beadded to the construct to support chondrogenesis.

In accordance with another aspect, there is provided a composition forregenerating cartilage comprising human mesenchymal stem cells and analginate gel. Preferably the matrix supports the differentiation andmaturation of human mesenchymal stem cells into chondrocytes.

In one embodiment, the construct can be placed in vitro in culture mediawhich will provide conditions favorable for chondrogenic differentiationof the MSCs in the gel. The constructs are cultured in this media andmay be modified to determine the effect of specific agents onchondrogenic differentiation and/or the chondrocytic phenotype.

In another embodiment, the MSCs are added to the gel in vitro underconditions such that the MSCs attach to the gel to form an MSC-gelconstruct. The construct can then be placed in vivo, i.e. implanted at atarget site. In this embodiment, the MSCs are not induced todifferentiate into chondrocytes prior to implantation. When placed invivo, the construct will be exposed to naturally occurring chondrogenicinducing factors, found for example in synovial fluid, to stimulatechondrogenic differentiation of the MSCs.

In a still further embodiment, the MSCs are added to the alginatesolution and the MSC-alginate solution is placed in contact withchondrogenic medium in vitro for a period of time sufficient to directthe MSCs into the chondrogenic lineage. The culture period may be longenough to obtain either mature chondrocytes or may be interrupted at anystage of the chondrogenic differentiation. The entire construct orportions thereof may then be implanted into the defect site.

In another aspect of this embodiment, the MSCs are added to the alginatesolution and the MSC alginate suspension is spread on a support. Thealginate suspension is contacted with a CaCl₂ solution. The alginatepolymerizes and forms a gel layer encasing the MSCs. The layer may thenbe contacted with a chondrogenesis inducing factor.

For purposes of the present invention, the MSCs can be culture-expandedMSCs, freshly isolated MSCs or unpurified populations of MSCs. The MSCsmay further be exposed to at least one chondroinductive agent.

Hyaluronic acid may be further added to the above embodiments to supportchondrogenesis.

The invention also provides a process for producing chondrocytes frommesenchymal stem cells by contacting mesenchymal stem cells with achondroinductive agent in vitro wherein the stem cells are associatedwith the alginate gel and then placed into the implant site.

The invention also provides a process for inducing chondrogenesis inmesenchymal stem cells by contacting mesenchymal stem cells with achondroinductive agent in vitro wherein the stem cells are associatedwith an alginate gel. The culture period may be long enough to obtaineither mature chondrocytes or may be interrupted at any stage of thechondrogenic differentiation. The entire construct or portion of theconstruct may be delivered to the defect site.

The invention further provides a method of repairing or regeneratingdamaged cartilage, comprising administering to an individual in needthereof a biocompatible construct comprising an alginate gel whichsupports the differentiation of human mesenchymal stem cells into thechondrogenic lineage.

The above methods can also preferably comprise steps where the cells arecultured with the chondroinductive composition and thereafter mixed inalginate gel suspension.

The above methods can further comprise steps where the cells arecultured with soluble hyaluronic acid and thereafter are mixed inalginate gel suspension.

In another embodiment of the present invention, the MSC-gel layer systemmay be delivered directly to the implant site without prior induction ofdifferentiation of the MSCs to the chondrogenic lineage. In thisembodiment the MSCs are allowed to attach to the gel for a period of upto 24 hours and then implanted without attempting to direct them intothe chondrogenic lineage prior to implantation.

In an alternate embodiment, the MSC-loaded gel is placed intochondrogenic medium in vitro for a finite period to direct the MSCs intothe chondrogenic lineage. The culture period may be long enough toobtain mature chondrocytes or may be interrupted at any stage of thechondrogenic differentiation. The entire construct or portions thereofmay be delivered to the defect site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an MSC-alginate-HA suspension layered onto themembrane surface of a transwell insert tissue culture well.

FIG. 2 illustrates the layer system of MSC-alginate-HA coated transwellin a tissue culture well with complete chondrogenic media andchondrogenesis inducing factor above and incomplete chondrogenic mediabelow the transmembrane layer.

FIG. 3 shows a cross section view of the alginate-MSC layer after 14days in culture after histological staining with toluidine-blue,safrinin-O and immunohistochemical staining of collagen Type II tissue.

FIG. 4. MSCs cultured under chondrogenic conditions in (A) alginatelayer and (B) pellets for up to 21 days and then stained for reactivitywith a collagen Type II-specific antibody, (C) Accumulated GAG and rateof biosynthesis measured after 14 days for both alginate and pelletcultures.

FIG. 5. Accumulation of GAG and rate of biosynthesis measured as afunction of concentration of added HA. The HA concentration ranted from50 to 1,000 μg/ml. Measurements were taken after 14 days in culture.

FIG. 6. Accumulation of GAG, DNA content, and rate of ³⁵S-sulfateincorporation in MSCs to which hyaluronan was or was not added.

FIG. 7. Rate of ³⁵S-sulfate incorporation in MSCs plated at differentinitial cell densities.

FIG. 8. Effect of HA on GAG synthesis at varying initial cell densitiesof MSCs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition for the repair of cartilagedefects by the rapid regeneration of cartilage tissue. The compositionis, for example, inserted or implanted into the defect resulting inarticular cartilage regeneration and repair of the defect.

The composition comprises an alginate layer in combination with isolatedmesenchymal stem cells. The alginate can be combined with the cartilageregenerative cells and optionally other active ingredients by forming asuspension of the MSCs and the alginate where the suspension liquid canhave other active ingredients dissolved.

Alginate is an unbranched linear polysaccharide consisting of β-Dmannuronic acid and α-L guluronic acid. It polymerizes and forms a gelin the presence of divalent cations such as Ca⁺⁺.

In one embodiment, the composition can contain additional componentssuch as chondroinductive factors. The cells and/or the alginate gel canbe contacted with a chondroinductive factor. As used herein the terms“chondroinductive agent” or “chondroinductive factor” refers to anynatural or synthetic, organic or inorganic chemical or biochemicalcompound or combination or mixture of compounds, or any mechanical orother physical device, container, influence or force that can be appliedto human mesenchymal stem cells which are in a three dimensional formatso as to effect their in vitro chondrogenic induction or the productionof chondrocytes. The chondroinductive agent is preferably selected,individually or in combination, from the group consisting of (i) aglucocorticoid, such as dexamethasone, and (ii) a member of thetransforming growth factor superfamily, such as a bone morphogenicprotein (preferably BMP-2 or BMP-4). TGF-β, inhibin A or chondrogenicstimulating activity factor (CSA).

The invention also provides a method for treating a cartilage detect inan animal, particularly a mammal, and more particularly a human in needthereof, which comprises administering to the cartilage defect of saidanimal a cartilage-regenerative amount of the composition of theinvention.

In one embodiment, the cells are contacted with a chondroinductivefactor while in the alginate gel layer ex vivo. Thus the method canfurther comprise administering at least one chondroinductive factorwhich further induces or accelerates the differentiation of suchmesenchymal stein cells into the chondrogenic lineage.

In another embodiment, the MSCs are first combined with the alginatesuspension and polymerized within the gel and are then implanted withoutinduction to chondrogenesis. In a still further embodiment. MSCs areloaded into the alginate and then placed in chondrogenesis-inducingmedia to induce differentiation prior to delivery to the defect site.

This invention has multiple uses and advantages. One such advantage liesin the ability to direct and accelerate MSC differentiation prior toimplantation into the host. For example, MSCs which are directed invitro to become chondrogenic cells will synthesize cartilage matrix atan implant site more rapidly and uniformly than MSCs which must first berecruited into the lineage and then progress through the keydifferentiation steps. Such an ex vivo treatment also provides foruniform and controlled application of bioactive factors to purifiedMSCs, leading to uniform lineage commitment and differentiation. Inaddition, by pretreating the MSCs prior to implantation, potentiallyharmful side effects associated with systemic or local administration ofexogenous bioactive factors are avoided. Another use of this techniquelies in the ability to direct tissue regeneration based on the stage ofdifferentiation of the cells at the time of implantation, which, withrespect to cartilage, may control the ultimate tissue type formed.

The cells are grown and maintained in a growth or culture medium inwhich the composition of the invention can undergo in vitrochondrogenesis, particularly in accordance with the methods of theinvention, such as serum-free animal cell culture preparation or mediumof known composition which will support the viability of humanmesenchymal stem cells in vitro.

The human mesenchymal stem cells utilized for purposes of the presentinvention can be derived, for example, from hone marrow. Although thesecells are normally present at very low frequencies in bone marrow, aprocess for isolating, and culture expanding the population of thesecells in tissue culture is reported in Caplan et al. U.S. Pat. No.5,486,359.

In one embodiment, the mesenchymal stem cells are preferably isolated,culture expanded human mesenchymal stem cells in a chemically definedserum-free medium which comprises (1) a chemically defined minimumessential medium (e.g., any of the Eagle's based media, i.e., Dulbecco'sModified Eagle's Medium (DMEM); Iscove's Modified Eagle's Medium, alphaModified Eagle's Medium, and also McCoy's 5A and BGJb (Fitton-JacksonModification)); (2) ascorbate or an analog thereof; (3) an iron source;(4) insulin or an insulin-like growth factor; and (5) at least onechondroinductive agent or factor.

It is also possible to use an isolated, non-cultured human mesenchymalstem cell preparation in the composition and methods of the invention.MSCs can be isolated as a non-cultured preparation, such as by densitygradient fractionation, from tissue such as bone marrow, blood(including peripheral blood), periosteum and dermis, and other tissueswhich have mesodermal origins, so as to be substantially free of othertypes of cells in the marrow. A monoclonal antibody separation is thenperformed as follows. Dynabeads M-450 (Dynal Inc., Lake Success, N.Y.)are coupled to anti-MSC monoclonal antibodies having ATCC AccessionNumbers HB 10743, HB 10744 and HB 10745, by incubating antibody withsecondary antibody coated Dynabeads (2.0 g anti-MSC antibody/mgDynabead; 1×10⁷ Dynabeads/ml) in PBS for 30 minutes at 4° C.

The cells are suspended in a solution of sodium alginate and thesuspension is distributed on a semiporous membrane as a layer, the shapeand dimensions of which can be easily modified. The alginate mixture issolidified by immersion of the layer and supporting membrane in a poolof calcium chloride.

In a preferred embodiment the alginate layer system comprises acomponent that provides for two separate media-tissue interfaces. Thishas the advantage of simulating the division of nutritional support seenin vivo between the subchondral vascular supply and the synovial fluidproximal to the articular surface. Nutritional or signalling gradientsmay be established by manipulating the media to formulations in eithermedia compartment.

Simple disks of material may be cast and cultured for use as an in vitrotesting platform permitting easier sample manipulations and multipleanalysis from the same sample. Biochemical, molecular and biomechanicalanalysis is possible from the same sample. The system is ideally suitedfor studying the effects of bioactive substances involved in modulatingthe differentiation of MSCs and represents a format for high throughputscreening of substances involved in the turnover of the extracellularmatrix molecules in cartilage under normal and osteoarthrtic conditions.

The cell density within the construct can easily be varied. Constructshape is defined by the dimensions and conformation of the supportingmembrane and may be further modified by progressive layering andsolidifying of the alginate mixture. Layering in this fashion could alsobe used to modify cellularity as a function of depth. Thicknessesapproaching articular cartilage (0.5-4.0 mm) are achievable. This leadsto possible uses as an implantable tissue construct for surgicalapplications. In this aspect, MSCs are culture expanded to appropriatenumbers, cast in an alginate layer, sized and shaped to fit anindividual's cartilage defect (or larger). The construct is culturedunder conditions which are conducive to chondrogenic differentiation.The newly formed chondrocytes express and organize extracellular matrixmolecules into a tissue which is comparable to articular cartilage inits responsiveness to physiological and biomechanical stresses. Within amatter of days of ex vivo culture, this tissue is surgically implantedor grafted into the site of the defect.

Whereas particular embodiments of the invention are described herein forpurposes of illustration, it will be evident to those skilled in the artthat numerous variations of the details may be made without departingfrom the invention as defined in the appended claims.

EXAMPLE 1

Mesenchymal stein cell were obtained from human bone marrows (PoeticTechnologies. Gaithersburg. Md.). Following normal expansion culture,4×10⁶ hMSCs were spun down and washed with 0.5 M NaCl. The cells wereresuspended in 10 μl sodium alginate (Monsanto, San Diego, Calif.) (2.4%(v/v) in 0.15 M NaCl, sterilized using a 0.45 μm filter) and 10 μlsoluble HA (HEALON (Pharmacia, Piseataway, N.J.) 2 μg/ml in sterile MQH₂O). This cell suspension was spread onto the membrane surface of aFALCON® transwell-insert tissue culture well (Becton-Dickinson, N.J.)(see FIG. 1). The transwell membrane (1.0 μm pore size) serves as asupport for the layer while the alginate polymerizes and forms a gelwhen the transwell is immersed in sterile 100 mM CaCl₂ for 10 minutes.Following solidification of the layer, the CaCl₂ as removed and thelayer with transwell was washed in 0.15M NaCl three times and twice withcomplete chondrogenic media (Table 1) containing 10 ng/ml recombinanthuman TGF-β3 (Oncogene Sciences. Cambridge. Mass.) was added above thelayer, incomplete chondrogenic media (Table 2) below (see FIG. 2).Culture of the layer proceeded for 14 days feeding twice per day due tothe high cell density. At the end of this period the tissue was fixedfor histological evaluation.

Histological staining revealed positive markers of chondrogenesisin >90% of the tissue. Uniform staining by toluidine blue and safranin-Oindicated sulfated proteoglycan production. Likewise, evenly distributedimmunohistochemical staining of collagen type II indicated acartilage-like tissue (see FIG. 3).

TABLE 1 Complete Chondrogenic Medium Ingredient Stock Dilution FinalConcentration DMEM as supplied n/a Undiluted (high glucose) ITS + assupplied 1:99 6.25 μg/ml bovine insulin supplement 6.25 μg/mltransferrin 6.25 μg/ml selenous acid 5.33 μg/ml linoleic acid 1.25 mg/mlBSA Dexamethasone¹ 1 mM in EtOH 2 serial  100 nM (FW = 392) 1:99 eachAscorbic acid-2- 5 mg/ml 1:99   50 μg/ml phosphate (FW = 290) (AA2P)²Proline² 4 mg/ml 1:99   40 μg/ml (FW = 115) Sodium pyruvate 100 mM 1:99  1 mM Antibiotic- as supplied 1:99  100 U/ml penicillin antimycotic 100 μg/ml streptomycin  250 ng/ml amphotericin B TGF-β3³ 5 μg/ml 1:500  10 ng/ml ¹Dexamethasone powder is dissolved in absolute ethanol (3.92mg per 10 ml), filter sterilized and stored at 4° C. ²Stocks of AA2P andproline are made by dissolving powder into DMEM and filter-sterilizing.Aliquots of these stocks may be frozen at −20° C. and stored for twoweeks. ³TGF-β3 stock is made by resuspending lyophilized powder insterile liquid using siliconized pipet tips and 0.5 ml tubes. 1 μg ofTGF-β3 is resuspended in 50 μl of 10% ethanol/10 mM HCl, then dividedinto aliquots and stored at −80° C. for up to 2 months.

TABLE 2 Incomplete Chondrogenic Medium Ingredient Stock Dilution FinalConcentration DMEM as supplied n/a Undiluted (high glucose) ITS + assupplied 1:99 6.25 μg/ml bovine insulin supplement 6.25 μg/mltransferrin 6.25 μg/ml selenous acid 5.33 μg/ml linoleic acid 1.25 mg/mlBSA Dexamethasone¹ 1 mM in EtOH 2 serial  100 nM (FW = 392) 1:99 eachAscorbic acid-2- 5 mg/ml 1:99   50 μg/ml phosphate (FW = 290) (AA2P)²Proline² 4 mg/ml 1:99   40 μg/ml (FW = 115) Sodium pyruvate 100 mM 1:99  1 mM Antibiotic- as supplied 1:99  100 U/ml penicillin antimycotic 100 μg/ml streptomycin  250 ng/ml amphotericin B ¹Dexamethasone powderis dissolved in absolute ethanol (3.92 mg per 10 ml), filter sterilizedand stored at 4° C. ²Stocks of AA2P and proline are made by dissolvingpowder into DMEM and filter-sterilizing. Aliquots of these stocks may befrozen at −20° C. and stored for two weeks.

EXAMPLE 2

Comparison of Chondrogenic Cultures of MSCs in Alginate Layers and inPellets

MSCs were isolated from human bone marrow and cultured underchondrogenic conditions in pellet format and in alginate layers. Sampleswere harvested for immunocytochemical detection of collagen Type II at7, 14 and 21 days. The cells were seeded on the alginate at a density of25×10⁶ cells/ml of alginate gel. Pellets were prepared using 2×10⁵cells/pellet. Within the alginate cultures deposition of collagen TypeII was evident at day 7 (FIG. 4A) and was distributed uniformilythroughout the inter-territorial matrix by day 14. After 21 days, thelayer noticeably as thicker with a dense extracellular matrix and thecells were similar morphologically to chondrocytes. These alginate layercultures were compared with pellet cultures of MSCs taken from the samedonor and grown under the same nutrient conditions. Immunocytochemicalanalysis of the pellet cultures (FIG. 4B) indicated that the depositionof collagen Type II occurred substantially later in the pellets comparedto the alginate culture. This is demonstrated clearly by comparing thelevel of collagen Type II staining in both cultures at 14 days (FIGS. 4Aand 4B). Furthermore, the cellular morphology was more uniform in thealginate cultures compared to the pellets. After 21 days underchondrogenic conditions, the cells in the alginate layer had ahomogeneous appearance with uniform staining while the pellets hadheterogeneous staining and cells of variable morphology. In particular,the outer periphery of the pellet had cells which were flattened and didnot express collagen Type II, an observation that has been madepreviously. In the alginate layer it appeared that all the cells,including those in the superficial zones, were differentiated, showingboth chondrocytic morphology and collagen Type II staining.

Analysis of the accumulation of sulfated glycosaminoglycans (GAG) wascarried out by measuring the amount of dimethylmethylene blue-reactivematerial in extracts of both alginate layers and pellets (FIG. 4C). Therate of GAG synthesis was determined by measuring the incorporation of³⁵S-sulfate at 14 days. Both measurements are given per ng DNA,indicating the level of activity per cell. The amount of accumulated GAGwas approximately 6-fold higher in the alginate culture compared to thepellet and the rate of GAG synthesis was about 2.5-fold higher.Incorporated ³⁵S-sulfate released into the culture media comprisedapproximately 10% and 4%, respectively, of the total GAG synthesized inpellet cultures and in alginate layers.

EXAMPLE 3

Effect of Addition of Hyaluronan (HA)

The effect of adding high molecular weight HA during the initialalginate layer formation was tested over a range of concentrations from50-1000 μg/ml (FIG. 5). GAG accumulation and synthesis at day 14 bothwere measured over the range of HA concentrations studied. Under theseconditions, the effect of added HA was greatest at 100 μg/ml HA.

To evaluate in more detail the manner in which HA influenced matrixdeposition, cultures were maintained over a longer time period with andwithout added HA. Cells were mixed with 250 μg/ml of HA at a seedingdensity of 25×10⁶ cells/ml. Samples were harvested at 7, 14 and 21 daysand the amount of GAG accumulated within the matrix, the DNA content andthe rate of ³⁵S-sulfate incorporation were measured (FIG. 6). After 7days in culture, the amount of GAG deposited in the matrix wasunaffected by the addition of HA. However, the rate of sulfateincorporation was enhanced at 3 and 7 days by the addition of HA. After14 days in culture, the rate of synthesis and the amount of accumulatedGAG were both enhanced by adding exogenous HA. At 21 days, the effect onaccumulation was not as great and there was no effect on sulfateincorporation. The DNA content was not affected significantly by theaddition of HA except at 3 days when it was reduced.

These results suggest that the influence that HA bears on MSCs is moreapparent when the cells already have initiated the process ofdifferentiation, and that early events appear not to be influenced. Thismight suggest that HA influences the deposition and assembly of anintegrated matrix, with a consequent feedback activation of proteoglycansynthesis.

EXAMPLE 4

Influence of Cell Density

Because chondrogenesis depends on contact between neighboring cells, itmay be expected that cell density would have an effect on the rate atwhich matrix was deposited. The alginate layer system offers the idealformat for evaluating factors such as cell density because the culturesare prepared as a suspension of cells in liquid alginate. In thisparticular experiment, cells were seeded into alginate layers atdensities ranging from 1.56 to 50×10⁶ cells/ml. Each layer was culturedfor 14 days under chondrogenic conditions and in the presence of TGF-β3.The rate of ³⁵S-sulfate incorporation was determined by adding labeledsulfate 24 hours prior to harvesting (FIG. 7). The DNA content was alsomeasured. Some clumping of cells was apparent at all cell densities. Atcell seeding densities of less than 6.25×10⁶ cells/ml, ³⁵S-sulfatelevels approached background after 14 days in culture (FIG. 7). However,a cell density of 6.25−25×10⁶ cells/ml showed increasing levels of GAGsynthesis. At a cell density of 50×10⁶ cells/ml, there was littleenhancement in biosynthesis compared to cultures at 25×10₆ cell/ml (FIG.7).

EXAMPLE 5

Hyaluronan Exerts a Greater Effect at Lower Cell Density

A study was carried out to assess ho GAG synthesis was affected atdifferent cell densities by HA addition. When cells were cultured inalginate at densities ranging from 3.2−25×10⁶ cells/ml with and withoutHA (FIG. 8), it was evident that (1) HA had a positive effect on GAGsynthesis at all densities and (2) the magnitude of the effect increasedas the cell density decreased. This suggested that HA, at least in part,overcame the inhibition caused by low cell-cell contact. Otherexperiments (not shown) indicated that at higher cell density (50×10⁶cells/ml) the addition of HA was without effect.

SUMMARY

These results indicate the following: (1) the rate of chondrogenicdifferentiation is enhanced when MSCs are cultured in alginate layersrather than pellets; (2) the rate of chondrogenesis is enhanced when HAis added at either 100 or 250 μg/ml to the culture medium; (3) the rateof chondrogenesis is enhanced when cultures in alginate are seeded withcells at a density 25×10⁶ cells/ml, and reduced at lower densities; (4)the positive effect of added HA is evident at all cell densities up to50×10⁶ cells/ml and the magnitude of the effect increases as the celldensity decreases.

What is claimed is:
 1. A composition for producing cartilage, comprisinghuman mesenchymal stem cells in an alginate gel layer which supports thedifferentiation and maturation of human mesenchymal stem cells intochondrocytes and hyaluronic acid, and wherein the mesenchymal stem cellsin the gel layer have been contacted with a chondroinductive agent. 2.The composition of claim 1 wherein said chondroinductive agent isselected from the group consisting of a glucocorticoid and a member ofthe transforming growth factor superfamily.
 3. The composition of claim2 wherein said chondroinductive factor is TGF-β3.
 4. The composition ofclaim 1 wherein said mesenchymal stem cells are in said alginate gellayer at a density from 3.2×10⁶ cells/ml to 25×10⁶ cells/ml.
 5. Thecomposition of claim 4 wherein said mesenchymal stem cells are in saidalginate gel layer at a density from 6.25×10⁶ cells/ml to 25×10⁶cells/ml.
 6. A method for regenerating or repairing cartilage in anindividual in need thereof comprising administering to said individualhuman a composition comprising mesenchymal stem cells in an alginate gellayer which supports the differentiation and maturation of humanmesenchymal stem cells into a chondrogenic lineage to an extentsufficient to accelerate cartilage formation therefrom and hyaluronicacid, and wherein the mesenchymal stem cells in the gel layer have beencontacted with a chondroinductive agent.
 7. The method of claim 6wherein said chondroinductive agent is selected from the groupconsisting of a glucocorticoid and a member of the transforming growthfactor superfamily.
 8. The method of claim 7 wherein saidchondroinductive agent is TGF-β3.
 9. The method of claim 6 wherein saidmesenchymal stem cells are in said alginate gel layer at a density from3.2×10⁶ cells/ml to 25×10⁶ cells/ml.
 10. The method of claim 9 whereinsaid mesenchymal stem cells are in said alginate gel layer at a densityfrom 6.25×10⁶ cells/ml to 25×10⁶ cells/ml.
 11. A method of formingcartilage in vitro, comprising: admixing human mesenchymal stem cellswith a solution comprising an alginate and hyaluronic acid; polymerizingsaid alginate to form a composition comprising said human mesenchymalstem cells in an alginate gel layer; and contacting said humanmesenchymal stem cells in the alginate gel layer with a chondroinductiveagent.
 12. The method of claim 11 wherein said alginate is sodiumalginate.
 13. The method of claim 11 wherein said chondroinductive agentis selected from the group consisting of a glucocorticoid and a memberof the transforming growth factor superfamily.
 14. The method of claim13 wherein said chondroinductive agent is TGF-β3.
 15. The method ofclaim 11 wherein said mesenchymal stem cells are in said alginate gellayer at a density from 3.2×10⁶ cells/ml to 25×10⁶ cells/ml.
 16. Themethod of claim 15 wherein said mesenchymal stem cells are in saidalginate gel layer at a density of from 6.25×10⁶ cells/ml to 25×10⁶cells/ml.